This disclosure relates to methods of detecting and/or quantifying hybridization of target molecules to microarrays.
Several of the more common strategies for the detection of hybridization between a single-stranded oligonucleotide probe and a complementary DNA target sequence include those that involve the use of tags with characteristic properties (such as fluorescence, color, luminescence or radioactivity), gold colloids or gold colloids enhanced with silver metal, and/or microscopy, surface enhanced Raman spectroscopy, surface plasmon resonance (SPR) spectroscopy, electrochemistry, quartz crystal microbalance, scanometry, digital cameras, or light scattering. However, only some of these techniques have been applied to the high throughput screening of DNA microarrays.
Fluorescent dyes have been used in conjunction with DNA microarray technology for gene expression profiling, analysis of single nucleotide polymorphisms, genotyping, biomarker discovery, clinical diagnostics, and other applications. However, fluorescent labels suffer from drawbacks such as broad overlapping emission peaks which limit multiplexing, quenching of fluorescence, and nonuniform fluorophore photobleaching. As such, alternative fluorophore-free strategies for DNA arrays are desirable.
As an alternative to the commonly used fluorescent labels for detection of DNA hybridization, the silver-augmented gold nanoparticle strategy was applied to DNA arrays for detection on gold films by SPR or glass slides by scanometry. Quantification of the extent of hybridization in scanometry was based on the intensity of the imaged grayscale observed for the darkened silver spots.
Infrared spectroscopy has been a valuable tool for the study of bioconjugate chemistry on substrates that can either transmit (ZnSe, CaF2) or reflect (gold, silver) IR light, and for the structural characterization of DNA bases and monolayers of oligonucleotide probes on gold surfaces. It was also used to qualitatively detect DNA hybridization between a single-stranded oligonucleotide probe, immobilized on the entire surface of a gold-coated slide, and its complementary synthetic target sequence. At this time, infrared microspectroscopy has not been applied to the detection of DNA microarrays primarily due to the lack of sensitivity of mercury cadmium telluride (MCT) infrared detectors (single point or focal plane array) for measuring trace amounts of biological material (DNA duplex) present in microarrayed 30 to 300 μm-diameter spots or larger on any IR substrate.
What is needed are additional fluorophore-free methods for the detection of target-capture probe hybrids on microarrays.